The overall objective of this research is to define the molecular mechanisms underlying the atrophy and death of neurons and the development of intraneuronal and extraneuronal lesion in Alzheimer's disease (AD). We have recently detected a population of slowly transported phosphoproteins in retinal ganglion cell neurons which comprise major microtubule-associated proteins (MAPS) and constituents of the fibrous and membrane cytoskeleton, including proteins with known relevance to AD. Major goals will be to use neuron specific in vivo approaches to identify these proteins and to define how phosphorylation may govern their turnover and interactions with cytoskeletal elements and the membrane. Our specific aims are to characterize the neuronal phosphoproteins with respect to associations with the Triton-insoluble cytoskeleton and ability to co-assemble with micro tubules. Their identities will be established immunochemically using well-character antibodies to known brain MAPs and cytoskeletal proteins and by 2-D SDS-PAGE and 2-D iodopeptide mapping. The number and relative locations of phosphorylated sites on the radiolabeled proteins in vivo will be determined by 2-D phosphopeptide mapping and compared with maps of the same polypeptide phosphorylated in vitro by each of four major protein kinases in order to identify the kinase(s) that are capable of mediating the vivo phosphorylation of specific sites and their relation to function. Site-specific phosphate turnover on proteins will be studied during axoplasmic transport in relation to changing associations of these polypeptides with specific moving and stationary cytoskeletal elements. Interrelationships between phosphorylation state, turnover rate and association with specific cytoskeletal organelles will be established. This information will be applied to additional investigations on the mechanism of amyloid accumulate AD brain. In separate experiments, the amyloid precursor protein of Alzheimer's disease will be studied with respect to its susceptibility to purified human brain proteins in vitro, its proteolytic cleavage patterns and its turnover and possible phosphorylation in cultured neural cells transfected with cDNA encoding the amyloid protein. By focusing on proteins of known or suspected importance in AD and on regulatory processes that govern cytoskeleton-membrane, these studies are expected to clarify the relationship between intraneuronal and extraneuronal lesions in Alzheimer's disease and will provide information directly relevant to the mechanism by which amyloid and tau proteins accumulate in AD brain.